The present invention relates to a method for isolating urea present in blood samples.
Urea is an organic compound, which constitutes an end product of the metabolism of nitrogen compounds in the human organism. In humans, urea is excreted with the urine.
Urea is primarily produced in liver cells and to a lesser extent in the kidneys. The production of urea in the body is associated with a variety of diseases, some of them congenital, which can cause significant damage to a person's health. Determining the production of urea is an indicator of the function of the liver, for example during liver transplants or the transplantation of liver cells.
For example, on page 213, Tuchman et al., Pediatric Research 2008 (64), describe a deficiency of N-acetylglutamate synthase and the analysis of urea production.
So as to measure the production of urea, patients were orally administered 13C-labeled sodium-acetate, which in the body resulted in the production of 13C-labeled urea; 13C-labeled acetate turns into 13CO2, which is converted to 13C carbamoyl-phosphate and then to 13C urea.
The majority of chemical elements exist in nature in form of mixtures of several stable or radioactive isotopes. Even in tracer studies using enriched compounds, isotopic abundance is generally indicated using the unit atom percent (atom %) or ppm. The relative delta scale in parts per thousand (‰) exists to describe the variations in the range of natural abundance. The δ values (such as δ13C, δ15 N, δ18O) are defined as the difference of the respective isotope ratio R ([heavy isotope]/[light isotope], for example R13C=[13C]/[12C]) of the sample compared to a standard, relative to this standard.
For example, the δ13C value is calculated as follows:
            δ      13        ⁢    C    =                                          R            sample                    -                      R            Standard                                    R          Standard                    ·      1000        =                  (                                            R              sample                                      R              Standard                                -          1                )            ·      1000      
The standard for carbon is limestone, Pee Dee Belemnite (PDB). The carbon that is bound by inserting CO2 in the photosynthesis is generally depleted of 13C. The majority of plants reduce CO2 to form carbohydrates according to the Calvin-Benson or C3 pathway. This causes the biomass of C3 plants (which include the useful plants rice, potatoes, soy, sugar beets, and cereals) to show δ13C values in the range of −24 to −32‰. Other plants fix CO2 according to the Hatch-Slack or C4 pathway. The δ13C values of products from C4 plants (corn, millet, and sugar cane) have δ13C values in the range of −10 to −16‰. This allows δ13C values to be used to check the origin and uniqueness of organic substances.
The δ13C value of plasma urea is usually determined by way of reacting urea to form CO2 using an enzyme. Therefore, it is important that the urea solution that is isolated from plasma is free of foreign CO2. However, CO2 is generally always present in the plasma, either as dissolved free CO2 or as bound CO2 in the form of bicarbonate. Freeing the plasma entirely from CO2 is not easy to do; in addition, CO2 must be prevented from being introduced in the sample during the isolation.
Tuchman et al. employ a method comprising the following steps to isolate the urea from blood plasma:
A plasma sample of 0.5 ml was mixed with 0.5 ml H2O and 40 μl 60% perchloric acid, and precipitated protein was separated. Thereafter, the container rested for 30 minutes so as to allow CO2 to be released. After transferring the mixture to a new vessel and adjusting the pH to the range of 6 to 7 using 300 μl KOH 1 M, precipitated potassium perchlorate was separated. The remaining bicarbonate was removed using an ion exchange column.
The column was washed with 1 ml HCl 10 mM and the eluate was dried in a glass container at 80° C. The sample rested overnight in a closed container in which also a piece of gauze, which was saturated with sodium hydroxide, was enclosed so as to absorb residues of CO2.
Thereafter, the container was rinsed with helium and the vessel was closed in an air-tight manner with a rubber stopper. An amount of 400 μl potassium phosphate buffer 0.5 M, pH 6.0, containing 3 mg urease enzyme/400 μl was injected through the rubber stopper. After one hour, 100 μl 20% phosphoric acid was added so as to release CO2 and stop the urease reaction. The released 13CO2 was measured using an isotope ratio mass spectrometer (IRMS).